Methods to predict and prevent resistance to taxoid compounds

ABSTRACT

Embodiments of the invention are directed to methods for predicting the resistance of cancer to members of the taxoid family by measuring the levels of prohibitin. Methods for treating cancer and taxoid family member resistant cancers using inhibitors of prohibitin, as well as therapeutic complexes that target prohibitin are also provided.

CROSS REFERENCE

This Application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/737,653 filed Nov. 17, 2005.

GOVERNMENT SUPPORT

This invention was supported, in part, by National Institutes of Health(NIH) Grant No. R37 CA37393. The government of the United States hascertain rights to the invention.

BACKGROUND OF THE INVENTION

Members of the taxoid family of compounds, e.g. docetaxel andpaclitaxel, have potent anti-tumor activities (Wang et al., Cancer88:2619, 2000, (5,6,7)). Docetaxel inhibits microtubule dynamics bybinding to beta-tubulin and blocking disassembly of alpha- andbeta-tubulin heterodimers thus abrogating tumor growth. Paclitaxel(Taxol™) is a complex diterepene derived from the Pacific yew tree Taxusbrevifolia (1) that also has significant anti-tumor activity. Paclitaxelprimarily suppresses microtubule dynamics and interferes with spindleformation arresting cell cycle at mitosis leading to apoptosis (6,7).

The clinical use of taxoid compounds has expanded to include cancers ofthe breast, ovaries and lung (2-4) and is expected to expand further. Aswith many cancer therapeutic agents, resistance to taxoid family membersremains a significant hindrance in their application as a successfulchemotherapeutic drugs. Resistance to the taxoid compounds can be eitherinherent or acquired subsequent to treatment most likely due toemergence of a minority population. For example, Paclitaxel resistanceis believed to be a multifactorial phenomenon. The principle mechanismsunderlying resistance include the overexpression of transporter proteinP-glycoprotein, altered binding of paclitaxel to its cellular target,β-tubulin, mutations in the β-tubulin gene, overexpression of β-tubulinisotypes, and decreased sensitivity to apoptotic stimuli. The role ofP-glycoprotein as a potential mediator of resistance has been abundantlystudied. Several P-glycoprotein inhibitors have been characterizedalthough relatively few of these, such as verapamil and cyclosporine,have shown any clinical efficacy and are frequently accompanied bydose-limiting side effects. Recently, there has been renewed effort tofind novel effectors of drug resistance which could provide alternativestrategies for resistance reversal.

SUMMARY OF INVENTION

We have undertaken a systematic proteomic approach to identify novelproteins associated with resistance to taxoid compounds (e.g. paclitaxeland docetaxel) and to examine their potential use as targets formodulating the resistant phenotype. We have identified prohibitin as aprotein that modulates sensitivity to the taxoid family memberpaclitaxel, as well as determined that multi-drug resistant cell lineshave elevated levels of prohibitin on their cell surface relative tocell lines that are sensitive to taxoid compounds. We have furtherconfirmed a role for GST-π in paclitaxel resistance (17) and demonstratethat it is possible to reverse paclitaxel resistance in vitro bysimultaneously silencing prohibitin and GST-π. Identification ofprohibitin as a modulator of resistance to taxoid family members hasimportant diagnostic and therapeutic implications for patients whosecancers are resistant to therapy with timid compounds.

Methods for predicting resistance of cancer to a member of the taxoidfamily in a subject are provided. In one embodiment, the level ofprohibitin in a biological sample obtained from the subject is measuredand compared to a standard level, wherein an elevation of the measuredlevel of prohibitin relative to the standard level is indicative ofresistance to a member of the taxoid family.

In one embodiment, a method for predicting the resistance of cancer in asubject to a member of the taxoid family is provided that comprisesmeasuring the level of prohibitin in multiple biological samplesobtained from a subject periodically over a period time; and measuring achange in the measured level of prohibitin in the biological samples. Anelevation of the measured level of prohibitin over time is indicative ofresistance to a member of the taxoid family.

Methods for improving the effectiveness of cancer treatment are alsoprovided. In one embodiment, the level of prohibitin in a biologicalsample is measured, wherein an elevated level of prohibitin as comparedto a standard level indicates that the subject is resistant to treatmentwith a member of the taxoid family and is in need of alternativetreatment thereby improving the effectiveness of cancer treatment.

In one embodiment, the biological sample is blood, tissue, serum,plasma, urine, stool, cerebrospinal fluid, nipple aspirates, tumorbiopsy, or cell lysate. In one embodiment, the subject has previouslybeen treated with a member of the taxoid family, e.g. paclitaxel ordocetaxel. In one embodiment, serial monitoring of the level ofprohibitin is performed at least quarterly, at least bimonthly, at leastbiweekly, at least weekly, at least every three days or at least daily.

In one embodiment, in methods of the invention, the level of prohibitinis measured by measuring the level of prohibitin on the cell surface ofcancer cells in the biological sample.

In one embodiment, in methods of the invention, the biological sample isa cell lysate and the level of prohibitin is measured by measuring thelevel of prohibitin in the microsomal fraction of the biological sample.

In one embodiment, in methods of the invention, the biological sample isserum and the level of prohibitin is determined by measuring the levelof prohibitin in the serum.

In one embodiment, in methods of the invention, the biological sample isblood and the level of prohibitin is determined by measuring the levelof prohibitin in platelets of the blood sample.

In one embodiment, in methods of the invention, the level of prohibitinprotein is measured using an antibody-based binding moiety whichspecifically binds prohibitin. The antibody based binding moiety can belabeled with a detectable label, for example a radioactive label, ahapten label, a fluorescent label, and an enzymatic label. In oneembodiment the antibody-based binding moiety is an antibody, e.g. amonoclonal antibody.

In one embodiment, in methods of the invention, the level of prohibitinprotein is measured using Western Blot analysis, Fluorescent activatedcell sorting (FACS), enzyme-linked immunosorbent assay (ELISA),immunohistochemistry, mass spectrometry, radio-immunoassy, surfaceplasmon resonance, or immunofluorescence.

Other embodiments of the invention provide methods for treating cancersthat are resistant to a taxoid family member.

In one embodiment, a method of treating a cancer is provided in whichthe subject is administered an inhibitor of prohibitin. In oneembodiment, the cancer is resistant to a taxoid family member. Inanother embodiment, the subject is administered a taxoid family member.In one embodiment, the taxoid family member is paclitaxel. In oneembodiment, the inhibitor of prohibitin inhibits translocation ofprohibitin to the cell surface. In another embodiment, the inhibitor ofprohibitin inhibits transcription or expression of prohibitin. In oneembodiment, the inhibitor of prohibitin is selected from an siRNA,antibody, small molecule, or peptide. In one embodiment, an inhibitor ofglutathione-S-transferase π and an inhibitor of prohibitin areadministered.

In another embodiment a method of treating a cancer that is resistant toa taxoid family member is provided. In the method, an agent thatselectively binds prohibitin is bound to a therapeutic agent andadministered to a subject.

In one embodiment, the therapeutic agent is a drug, a chemotherapeuticagent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent,a hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a peptide,a protein, an antibiotic, an antibody, a Fab fragment of an antibody, ahormone antagonist, or an antigen.

In one embodiment, the agent that selectively binds prohibitin is apeptide baying the amino acid sequence of CKGGEAKDC (SEQ ID NO:2).

In one embodiment, the agent is an antibody-based binding moiety.

Articles of manufacture comprising the prohibitin inhibitors andtherapeutic agents of the invention are also provided.

In other aspects of the invention, methods to direct treatment of asubject are provided. In one embodiment, the method comprises having asubject tested for the level of prohibitin in a biological sample,wherein a clinician reviews the results as compared to a standard levelof prohibitin, and if the biological sample has an elevated level ofprohibitin as compared to a standard level the clinician directs thesubject to be treated with a compound comprising an agent thatselectively binds prohibitin and a therapeutic agent The test may beperformed in the same country where the subject resides or in anothercountry and the results are made available, for example via a Web site,or are transmitted to the clinician.

In another embodiment, the method comprises having a subject tested forthe level of prohibitin in a biological sample, wherein a clinicianreviews the results as compared to a standard level of prohibitin, andif the biological sample has an elevated level of prohibitin as comparedto a standard level the clinician directs the subject to be treated witha inhibitor of prohibitin. The test may be performed in the same countrywhere the subject resides or in another country and the results are madeavailable, for example via a Web site or are transmitted to theclinician.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show graphs illustrating the effect of paclitaxel oncell proliferation. A549 and A549 TR cell lines (FIG. 1A) and MES-SA andMES-SA-DX5 cell lines (FIG. 1B) were treated with various concentrationsof paclitaxel. 24 hours after 1×10⁴ cells/well were seeded in 96 wellplates. MTT assay for cell survival was performed 72 h after paclitaxeladdition. X axis: concentration of paclitaxel Y axix-% survival.

FIGS. 2A to 2D show Western blot analysis of protein fractions and wholecell lysates. FIG. 2A, Western blot on the cytoplasmic fractions ofMES-SA and MES-SA DX5 (left panel) and A549 and A549TR (right panel)using GST-π antibodies. FIG. 28, Western blot on microsomal fractions ofMES-SA and MES-SA DX5 (left panel) and A549 and A549TR (right panel)using prohibitin antibodies. FIG. 2C, Western blot on whole cell lysatesof MES-SA and MES-SA DX5 (left panel) and A549 and A549TR (right panel)using GST-π antibodies. FIG. 2D, Western blot on whole cell lysates ofMES-SA and MES-SA DX5 (left panel) and A549 and A549TR (right panel)using prohibitin antibodies. Equivalent loading was demonstrated usingGAPDH and actin. Protein samples were prepared as described in thetwo-dimensional electrophoresis studies.

FIGS. 3A and 3B show bar graphs depicting the effect of silencing GST-πand prohibitin individually on paclitaxel sensitivity. FIG. 3A, GST-πsiRNA (Smartpool), and FIG. 3B, Prohibitin siRNA (Smartpool) transfectedA549-TR cells were treated with or without Paclitaxel 24 h aftertransfection. Cell survival was analyzed after 72 h of Paclitaxeltreatment by cell counting. Proteins were extracted for western blot atthe same time. Percent cell survival was calculated as percentage ofcell survival in GST-π and prohibitin siRNA transfected cells comparedto cells transfected by a negative control siRNA. (Inset): Western blotanalysis confirms knockdown of respective protein at the end of theassay time (96 h). Each bar is SD from a representative experiment donein at least in duplicates.

FIGS. 4A and 4B how bar graphs depicting the effect of combinedsilencing of GST-π and prohibitin using siRNA on paclitaxel sensitivityof resistant cells. FIG. 4A, A549TR cells and FIG. 4B, MES-SA DX5 cellswere transfected with siRNA for GST-π and prohibitin in combination, 24h after cells were seeded. Cells were then treated with or withoutPaclitaxel 24 h after siRNA transfection. Cell survival was analyzedafter 72 h of paclitaxel treatment by cell counting. Proteins wereextracted for Western blotting at the same time. Percent cell survivalwas calculated by comparing number of cells in GST-π and prohibitinsiRNA transfected cells compared to number of cells transfected by anegative control siRNA. Each bar represents the SD from a representativeexperiment performed at least in duplicates.

FIG. 5 shows the protein sequence of Homo Sapiens prohibitin (SEQ IDNO:1).

FIG. 6 shows the nucleic acid sequence of Homo Sapiens prohibitin (SEQID NO:7),

DESCRIPTION OF THE INVENTION

Embodiments of the present invention are directed to methods forpredicting the resistance of cancer to members of the taxoid family bymeasuring the levels of prohibitin. Methods for treating cancer andtaxoid family member resistant cancers are also provided.

As used herein, the term “a taxoid family member” or “taxoid compound”refers to a class of chemotherapeutic compounds belonging to the taxanefamily. Specific members of the taxoid family include, but are notlimited, to paclitaxel (Taxol™) , docetaxel (Taxotere™) and analogsthereof (i.e., XRP9881 and XRP6258; see Ojima and Geney, Curr OpinInvestig Drugs 4:73 7, 2004). This class of molecules are β-tubulinbinders and stabilize the polymerized form of the microtubule. “A taxoidfamily member resistant cancer” refers to a cancer that is not sensitiveto the taxoid family compounds, i.e. the cancer does not respond to theanti-cancer effects of the compounds.

As used herein, a “cancer” in a subject or patient refers to thepresence of cells possessing characteristics typical of cancer-causingcells, such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Often, cancer cells will be inthe form of a tumor, but such cells may exist alone within a subject, ormay be a non-tumorigenic cancer cell, such as a leukemia cell. In somecircumstances, cancer cells will be in the form of a tumor; such cellsmay exist locally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells. Examples of cancerinclude but are not limited to breast cancer, a melanoma, adrenal glandcancer, biliary tract cancer, bladder cancer, brain or central nervoussystem cancer, bronchus cancer, blastoma, carcinoma, a chondrosarcoma,cancer of the oral cavity or pharynx, cervical cancer, colon cancer,colorectal cancer, esophageal cancer, gastrointestinal cancer,glioblastoma, hepatic carcinoma, hepatoma, kidney cancer, leukemia,liver cancer, lung cancer, lymphoma, non-small cell lung cancer,osteosarcoma, ovarian cancer, pancreas cancer, peripheral nervous systemcancer, prostate cancer, sarcoma, salivary gland cancer, small bowel orappendix cancer, small-cell lung cancer, squamous cell cancer, stomachcancer, testis cancer, thyroid cancer, urinary bladder cancer, uterineor endometrial cancer, and vulval cancer. In one embodiment, the canceris not a lipoma.

As used herein, a “biological sample” refers to a sample of biologicalmaterial obtained from a patient, preferably a human patient, includinga tissue, a tissue sample, a cell sample, e.g., a tissue biopsy, suchas, an aspiration biopsy, a brush biopsy, a surface biopsy, a needlebiopsy, a punch biopsy, an excision biopsy, an open biopsy, an incisionbiopsy or an endoscopic biopsy), and a tumor sample. Biological samplescan also be biological fluid samples, including but not limited to,urine, blood, sawn, platelets, saliva, cerebrospinal fluid, nippleaspirates, and cell lysate (e.g. supernatant of whole cell lysate,microsomal fraction, membrane fraction, or cytoplasmic fraction). Thesample may be obtained using any methodology known to one skilled in theart. Methods for platelet extraction are found in PCT publication WO2005/103281.

Embodiments of the invention also encompasses the use of isolates of abiological sample in the methods of the invention. As used herein, an“isolate” of a biological sample (e. g., an isolate of a tissue or tumorsample) refers to a material or composition (e.g., a biological materialor composition) which has been separated, derived, extracted, purifiedor isolated from the sample and preferably is substantially free ofundesirable compositions and/or impurities or contaminants associatedwith the biological sample.

As used herein, a “tissue sample” refers to a portion, piece, part,segment, or fraction of a tissue which is obtained or removed from anintact tissue of a subject, preferably a human subject.

In one embodiment, the biological sample is treated as to preventdegradation of protein. Methods for inhibiting or preventing degradationinclude, but are not limited to, treatment of the biological sample withprotease, freezing the biological sample, or placing the biologicalsample on ice. Preferably, prior to analysis, the biological samples orisolates are constantly kept under conditions as to prevent degradationof protein, e.g. prohibitin.

As used herein “serially monitoring” a level of prohibitin in a sample,refers to measuring a level of prohibitin in a sample more than once,e.g., quarterly, bimonthly, monthly, biweekly, weekly, every three daysor daily. Serial monitoring of a level includes periodically measuring alevel of prohibitin at regular intervals as deemed necessary by theskilled artisan.

A used herein, the term “subject” or “patient” refers generally to amammal.

As used herein, “Prohibitin” refers to the Prohibitin protein ofGenebank accession NP_(—)002625 (Homosapiens) (SEQ ID NO:1) (FIG. 5).The term also encompasses species variants, homologues, allelic forms,mutant forms, and equivalents thereof.

The term “antagonist” or “inhibitor” is used in the broadest sense, andincludes any molecule that partially or fully blocks, inhibits, orneutralizes a biological activity of prohibitin or the transcription ortranslation thereof. Suitable antagonist molecules specifically includeantagonist antibodies or antibody fragments, fragments, peptides, smallorganic molecules, anti-sense nucleic acids, siRNA, etc.

The term “standard level” as used herein refers to a baseline amount ofprohibitin as determined in one or more normal subjects that does nothave a cancer resistant to a taxoid family member. For example, abaseline may be obtained from at least one subject and preferably isobtained from an average of subjects (e.g., n=2 to 100 or more), whereinthe subject or subjects have no prior history of resistance to a taxoidfamily member. A baseline standard level can also be derived from taxoidfamily member sensitive cell lines, a cell that is not resistant to ataxoid family member.

As used herein, the term “standard level” is also intended to include abaseline amount of prohibitin as determined in the subject that is to bemonitored for taxoid family member resistance. For example, one need notdirectly compare the amount of prohibitin in a subject's sample to astandard level derived from normal subjects, rather one can measure achange in concentration of prohibitin present in multiple biologicalsamples obtained from the subject over a period of time, e.g. thestandard level used for comparison is the level of prohibitin measuredin the first biological sample obtained from the subject. An elevationin the measured concentration of prohibitin over a period of time isindicative of resistance to a member of the taxoid family.

As used herein, “a period of time” is intended to include a period ofdays, weeks, months or even years. Multiple biological samples areobtained from a subject over a period of time, i.e. a biological sampleis obtained from a subject periodically over time at various intervals.A biological sample can be obtained from a subject at any interval, Forexample, a biological sample can be taken every day for weeks, months oryears. Alternatively, a biological sample can be obtained once a week,twice a week, three times a week, four times a week, five times a week,or six times a week for a period of weeks, months or years. In oneembodiment, a biological sample is obtained once a week over a period ofthree months. In one embodiment, a biological sample is obtained once amonth for a period of months, or years.

For purposes of comparison, the level of prohibitin in a biologicalsample to be measured is of the same type (obtained from the samebiological source) as what is used for determination of the baselinestandard level. For example, in one embodiment of the invention, thelevel of prohibitin is measured by measuring the level of prohibitin onthe cell surface of cancer cells in the biological sample. Thus, thebaseline standard level is determined by measuring the level ofprohibitin on the cell surface of non-resistant cancer cells.Alternatively, the biological sample is a cell lysate and the level ofprohibitin is measured by measuring the level of prohibitin in themicrosomal fraction of the biological sample. Thus, the baselinestandard level is determined by measuring the level of prohibitin in themicrosomal fraction of the biological sample obtained from non-resistantcancer cells. Means for isolation of the microsomal fraction from cellsare well known to those skilled in the art.

In one embodiment, the biological sample is serum and the level ofprohibitin is determined by measuring the level of prohibitin in theserum. Thus, the baseline standard level is determined by measuring thelevel of prohibitin in serum from a subject that does not have a taxoidfamily member resistant cancer.

In one embodiment, the biological sample is blood and the level ofprohibitin is determined by measuring the level of prohibitin inplatelets of the blood sample. Thus, the baseline standard level isdetermined by measuring the level of prohibitin in platelets from asubject that does not have a taxoid family member resistant cancer.

As used herein, “elevation” of a measured level of prohibitin relativeto a standard level means that the amount or concentration of prohibitinin a sample is sufficiently greater in a subject's biological samplerelative to the standard level of prohibitin. For example, elevation ofthe measured level relative to a standard level may be any statisticallysignificant elevation which is detectable. Such an elevation mayinclude, but is not limited to, about a 1%, about a 10%, about a 20%,about a 40%, about an 80%, about a 2-fold, about a 4-fold, about an8-fold, about a 20-fold, or about a 100-fold elevation, or more,relative to the standard. The term “about” as used herein, refers to anumerical value plus or minus 10% of the numerical value.

In one embodiment, the methods of the invention may be performedconcurrently with methods of detection for other analytes, e.g.,detection of mRNA or protein of other markers associated with taxoidfamily resistance (e.g. P-glycoprotein, β-tubulin, mutations in theβ-tubular gene, or overexpression of β-tubulin isotypes).

Measuring Levels of Prohibitin

The level of prohibitin protein can be measured by any means known toone skilled in the art including, but not limited to, competitionbinding assays, mass spectrometry, Western blot, fluorescent activatedcell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), antibodyarrays, high pressure liquid chromatography, optical biosensors, andsurface plasmon resonance.

In one embodiment, prohibitin protein is detected by contacting thebiological sample with an antibody-based binding moiety thatspecifically binds to prohibitin, or to a fragment of that protein.Formation of the antibody-protein complex is then detected and measuredto indicate protein levels. Anti-prohibitin antibodies are availablecommercially (e.g. human prohibitin affinity purified polyclonal andmonoclonal Ab's from R&D Systems, Inc. Minneapolis, Minn. 55413; AVIVASystems Biology, San Diego, Calif. 92121; see also U.S. Pat. No.5,463,026 Antibodies Alternatively, antibodies can be raised against thefull length prohibitin, or a portion of prohibits.

Antibodies for use in the present invention can be produced usingstandard methods to produce antibodies, for example, by monoclonalantibody production (Campbell, A. M., Monoclonal Antibodies Technology:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, the Netherlands (1984); St. Groth et al,J. Immunology, (1990) 35: 1-21; and Kozbor et al., Immunology Today(1983) 4:72). Antibodies can also be readily obtained by using antigenicportions of the protein to screen an antibody library, such as a phagedisplay library by methods well known in the art. For example, U.S. Pat.No. 5,702,892 (U.S.A. Health & Human Services) and WO 01/18058(Novopharm Biotech Inc.) disclose bacteriophage display libraries andselection methods for producing antibody binding domain fragments.

The term “antibody-based binding moiety” or “antibody” includesimmunoglobulin molecules and immunologically active determinants ofimmunoglobulin molecules, e.g., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) prohibitin.The term “antibody-based binding moiety” is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments thereof which are also specifically reactive with prohibitin,or fragments thereof. Antibodies can be fragmented using conventionaltechniques. Thus, the term includes segments of proteolytically-cleavedor recombinantly-prepared portions of an antibody molecule that arecapable of selectively reacting with a certain protein. Non limitingexamples of such proteolytic and/or recombinant fragments include Fab,F(ab′)2, Fab′ , Fv, dAbs and single chain antibodies (scFv) containing aVL and VH domain joined by a peptide linker. The scFv's may becovalently or non-covalently linked to form antibodies having two ormore binding sites. Thus, “antibody-base binding moiety” includespolyclonal, monoclonal, or other purified promotions of antibodies andrecombinant antibodies. The term “antibody-base binding moiety” isfurther intended to include humanized antibodies, bispecific antibodies,and chimeric molecules having at least one antigen binding determinantderived from an antibody molecule. In a preferred embodiment, theantibody-based binding moiety detectably labeled.

“Labeled antibody”, as used herein, includes antibodies that are labeledby a detectable means and include, but are not limited to, antibodiesthat are enzymatically, radioactively, fluorescently, andchemiluminescently labeled. Antibodies can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS.

In the methods of the invention that use antibody based binding moietiesfor the detection of profilin, the level of the protein of interestpresent in the biological samples correlate to the intensity of thesignal emitted from the detectably labeled antibody.

In one preferred embodiment, the antibody-based binding moiety isdetectably labeled by linking the antibody to an enzyme. The enzyme, inturn, when exposed to it's substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means.Non-limiting examples of enzymes which can be used to detectably labelthe antibodies of the present invention include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. Chemiluminescence is anothermethod that can be used to detect an antibody-based binding moiety.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling an antibody, it ispossible to detect the antibody through the use of radioimmune assays.The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by audoradiography. Isotopeswhich are particularly useful for the purpose of the present inventionare ³H, ¹³¹I, ³⁵S, ¹⁴C, and preferably ¹²⁵I.

It is also possible to label an antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are CYE dyes, fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

An antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The levels of prohibitin, in the biological samples, can be measured byimmunoassays, such as enzyme linked immunoabsorbent assay (ELISA),radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Westernblotting, or immunohistochemistry, each of which are described in moredetail below. In one embodiment, the presence of cell surface prohibitinis measured using FACS analysis, competition binding assays, orRIA/IRMA. Antibody arrays or protein chips can also be employed, see forexample U.S. Patent Application Nos: 20030013208A1; 20020155493A1;20030017515 and U.S. Pat. Nos. 6,329,209; 6,365,418, which are hereinincorporated by reference in their entirety.

Immunoassays

“Radioimmunoassay (RIA)” is a technique for detecting and measuring theconcentration of an antigen using a labeled, e.g.. radioactivelylabeled, form of the antigen. Examples of radioactive labels forantigens include ³H, ¹⁴C, and ¹²⁵I. The concentration of antigen, e.g.,prohibiting in a biological sample is measured by having the antigen inthe biological sample compete with the labeled, e.g. radioactively,antigen for binding to an antibody to the antigen. To ensure competitivebinding between the labeled antigen and the unlabeled antigen, thelabeled antigen is present In a concentration sufficient to saturate thebinding sites of the antibody. The higher the concentration of antigenin the sample, the lower the concentration of labeled antigen that willbind to the antibody.

In a radioimmunoassay, to determine the concentration of labeled antigenbound to antibody, the antigen-antibody complex must be separated fromthe free antigen. One method for separating the antigen-antibody complexfrom the free antigen is by precipitating the antigen-antibody complexwith an anti-isotype antiserum. Another method for separating theantigen-antibody complex from the free antigen is by precipitating theantigen-antibody complex with formalin-killed S. aureus. Yet anothermethod for separating the antigen-antibody complex from the free antigenis by performing a “solid-phase radioimmunoassay” where the antibody islinked (e.g., covalently) to Sepharose beads, polystyrene wells,polyvinylchloricie wells, or microtiter wells. By comparing theconcentration of labeled antigen bound to antibody to a standard curvebased on samples having a known concentration of antigen, theconcentration of antigen in the biological sample can be determined.

An “Immunoradiometric assay” (IRMA) is an immunoassay in which theantibody reagent is radioactively labeled. An IRMA requires theproduction of a multivalent antigen conjugate, by techniques such asconjugation to a protein e.g., rabbit serum albumin (RSA). Themultivalent antigen conjugate must have at least 2 antigen residues permolecule and the antigen residues must be of sufficient distance apartto allow binding by at least two antibodies to the antigen. For example,in an IRMA the multivalent antigen conjugate can be attached to a solidsurface such as a plastic sphere. Unlabeled “sample” antigen andantibody to antigen which is radioactively labeled are added to a testtube containing the multivalent antigen conjugate coated sphere. Theantigen in the sample competes with the multivalent antigen conjugatefor antigen antibody binding sites. Amer an appropriate incubationperiod, the unbound reactants are removed by washing and the amount ofradioactivity on the solid phase is determined. The amount of boundradioactive antibody is inversely proportional to the concentration ofantigen in the sample.

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA).” ELISA is a technique for detecting and measuring theconcentration of an antigen using a labeled, e.g., enzyme linked, formof the antibody. There are different forms of ELISA, which arc wellknown to those skilled in the art. The standard techniques known in theart for ELISA are described in “Methods in Immunodiagnosis”, 2ndEdition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell etal., “Methods and Immunology”, W. A. Benjamin, Inc., 1964; andOellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

In a “sandwich ELISA”, an antibody, e.g. antibodies against prohibitin,is linked to a solid phase, i.e. a microliter plate, and exposed to abiological sample containing antigen, prohibitin The solid phase is thenwashed to remove unbound antigen. A labeled antibody, e.g. enzymelinked, is then bound to the bound-antigen, if present, forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphates, horseradish peroxidase,luciferase, urease, and B-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured.

In a “competitive ELISA”, antibody is incubated with a sample containingantigen, e.g., prohibitin. The antigen-antibody mixture is thencontacted with a solid phase, e.g. a microliter plate, that is coatedwith antigen, e.g., prohibitin. The more antigen present in the sample,the less free antibody that will be available to bind to the solidphase. A labeled, e.g., enzyme linked, secondary antibody is then addedto the solid phase to determine the amount of primary antibody bound tothe solid phase.

Alternatively, prohibitin levels in cells and/or tumors can be detectedin vivo in a subject by introducing into the subject a labeled antibodyto prohibitin protein. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, immunohistochemistry (“IHC”) and immunocytochemistry(“ICC”) techniques are used. IHC is the application of immunochemistryto tissue sections, whereas ICC is the application of immunochemistry tocells or tissue imprints after they have undergone specific cytologicalpreparations such as, for example, livid-based preparations.Immunochemistry is a family of techniques based on the use of a specificantibody, wherein antibodies are used to specifically target moleculesinside or on the surface of cells. The antibody typically contains amarker that will undergo a biochemical reaction, and thereby experiencea change color, upon encountering the targeted molecules. In someinstances, signal amplification may be integrated into the particularprotocol, wherein a secondary antibody, that includes the marker stain,follows the application of a primary specific antibody.Immunoshistochernical assays are known to those of skill in the art(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985);Jalkanen, et al., J. Cell. Biol. 105;3087-3096 (1987).

Typically, for immunohistochemistry, tissue sections are obtained from apatient and fixed by a suitable fixing agent such as alcohol, acetone,and paraformaldehyde, to which is reacted an antibody. Conventionalmethods for immunohistochemistry are described in Harlow and Lane (eds)(1988) In “Antibodies A Laboratory Manual”, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.; Ausbel et al (eds) (1987), in CurrentProtocols In Molecular Biology, John Wiley and Sons (New York, N.Y.).Biological samples appropriate for such detection assays include, butare not limited to, cells, tissue biopsy, whole blood, plasma, serum,sputum, cerebrospinal fluid, pleural fluid, urine and the like.

For direct labeling techniques, a labeled antibody is used. For indirectlabeling techniques, the sample is further reacted with a labeledsubstance.

Alternatively, immunocytochemistry may be used. In general, cells areobtained from a patient and fixed by a suitable fixing agent such asalcohol, acetone, and paraformaldehyde, to which is reacted an antibody.Methods of immunocytological staining of human samples is known to thoseof skill in the art and described, for example, in Brauer et al., 2001(FASEB J, 15, 2689- 2701), Smith-Swintosky et al., 1997.

Immunological methods of the present invention are advantageous becausethey require only small quantities of biological material. Such methodsmay be done at the cellular level and thereby necessitate a minimum ofone cell. Preferably, several cells are obtained from a subject.

Other techniques may be used to detect the levels of prohibitinaccording to a practitioner's preference, based upon the presentdisclosure. One such technique is Western blotting (Towbin et al., Proc.Nat Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is runon an SOS-PAGE gel before being transferred to a solid support, such asa nitrocellulose filter. In one embodiment, western blotting is used todetect levels of prohibitin in the microsomal fraction of cell lysate.Detectably labeled antibodies can then be used to detect and/or assesslevels of the prohibitin protein where the intensity of the signal fromthe detectable label corresponds to the amount of protein. Levels can bequantitated, for example by densitometry.

Mass Spectometry

In addition, prohibitin levels may be detected using Mass Spectrometrysuch as MALDI/TOF (time-of-flight), SELDI/TOF, liquidchromatography-mass spectrometry (LC-MS), gas chromatography-massspectrometry (GC-MS), high performance liquid chromatography-massspectromeny (HPLC-MS), capillary electrophoresis-mass spectrometry,nuclear magnetic resonance spectrometry, or tandem mass spectrometry(e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. PatentApplication Nos: 20030199001, 20030134304, 20030077616, which are hereinincorporated by reference.

Mass spectrometry methods are well known in the art and have been usedto quantify and/or identify biomolecules, such as proteins (see, e.g.,Li et al. (2000) Tibtech 18:151-160; Rowley et al (2000) Methods 20:383-397; and Kuster and Mann (1998) Cum Opin. Structural Biol. 8:393-400). Further, mass spectrometric techniques have been developedthat permit at least partial de novo sequencing of isolated proteins.Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad.Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).

In certain embodiments, a gas phase ion spectrophotometer is used. Inother embodiments, laser-desorption/ionization mass spectrometry is usedto analyze the sample. Modern laser desorption/ionization massspectrometry (“LDI-MS”) can be practiced in two main variations: matrixassisted laser desorption/ionization (“MALDI”) mass spectrometry andsurface-enhanced laser desorption/ionization (“SELDI”). In MALDI, theanalyte is mixed with a solution containing a matrix, and a drop of theliquid is placed on the surface of a substrate. The matrix solution thenco-crystallizes with the biological molecules. The substrate is insertedinto the mass spectrometer. Laser energy is directed to the substratesurface where it desorbs and ionizes the biological molecules withoutsignificantly fragmenting them. However, MALDI has limitations as ananalytical toot. It does not provide means for fractionating the sample,and the matrix material can interfere with detection, especially for lowmolecular weight analytes. See, e.g., U.S. Pat. No. 5,118,937(Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

In SELDI, the substrate surface is modified so that it is an activeparticipant in the desorption process. In one variant, the surface isderivatized with adsorbent and/or capture reagents that selectively bindthe protein of interest In another variant, the surface is derivatizedwith energy absorbing molecules that are not desorbed when struck withthe laser. In another variant, the surface is derivatized with moleculesthat bind the protein of interest and that contain a photolytic bondthat is broken upon application of the laser. In each of these methods,the derivatizing agent generally is localized to a specific location onthe substrate surface where the sample is applied. See, e.g., U.S. Pat.No. 5,719,060 and WO 98159361. The two methods can be combined by, forexample, using a SELDI affinity surface to capture an analyte and addingmatrix-containing liquid to the captured analyte to provide the energyabsorbing material.

For additional information regarding mass spectrometers, see, e.g.,Principles of Instrumental Analysis, 3rd edition., Skoog, SaundersCollege Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia ofChemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York1995), pp. 1071-1094.

Detection of the presence of a marker or other substances will typicallyinvolve detection of signal Intensity. This, in turn, can reflect thequantity and character of a polypeptide bound to the substrate. Forexample, in certain embodiments, the signal strength of peak values fromspectra of a first sample and a second sample can be compared (e.g.,visually, by computer analysis etc.), to determine the relative amountsof particular biomolecules. Software programs such as the BiomarkerWizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be usedto aid in analyzing mass spectra. The mass spectrometers and theirtechniques are well known to those of skill in the art.

Any person skilled in the art understands, any of the components of amass spectrometer, e.g., desorption source, mass analyzer, detect, etc.,and varied sample preparations can be combined with other suitablecomponents or preparations described herein, or to those known in theart. For example, in some embodiments a control sample may contain heavyatoms, e.g. ¹³C, thereby permitting the test sample to mixed with theknown control sample in the same mass spectrometry run.

In one preferred embodiment, a laser desorption time-of-flight (TM massspectrometer is used. In laser desorption mass spectrometry, a substratewith a bound marker is introduced into an inlet system. The marker isdesorbed and ionized into the gas phase by laser from the ionizationsource. The ions generated are collected by an ion optic assembly, andthen in a lime-of-flight mass analyzer, ions are accelerated through ashort high voltage field and let drift into a high vacuum chamber. Atthe far end of the high vacuum chamber, the accelerated ions strike asensitive detector surface at a different time. Since the time-of-flightis a function of the mass of the ions, the elapsed time between ionformation and ion detector impact can be used to identify the presenceor absence of molecules of specific mass to charge ratio.

In some embodiments the relative amounts of one or more biomoleculespresent in a first or second sample is determined, in part, by executingan algorithm with a programmable digital computer, The algorithmidentifies at least one peak value in the first mass spectrum and thesecond mass spectrum. The algorithm then compares the signal strength ofthe peak value of the first mass spectrum to the signal strength of thepeak value of the second mass spectrum of the mass spectrum. Therelative signal strengths are an indication of the amount of thebiomolecule that is present in the first and second samples. A standardcontaining a known amount of a biomolecule can be analyzed as the secondsample to provide better quantify the amount of the biomolecule presentin the first sample. In certain embodiments, the identity of thebiomolecules in the first and second sample can also be determined.

In one embodiment of the invention, levels of prohibitin are detected byMALDI-TOF mass spectrometry.

Methods of detecting prohibitin in a biological samples also include theuse of surface plasmon resonance (SPR). In such assays an antibody thebinds prohibitin need not be detectably labeled and can be used withouta second antibody that binds to the specific polypeptide. For example,as antibody specific for prohibitin may be bound to an appropriate solidsubstrate and then exposed to the sample. Binding of a prohibitin to theantibody on the solid substrate may be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).Optical biosensors are also contemplated for use in embodiments of theinvention.

The SPR biosensing technology has been combined with MALDI-TOF massspectrometry for the desorption and identification of biomolecules. In achip-based approach to BIA-MS, a ligand, e.g., prohibitin antibody, iscovalently immobilized on the surface of a chip. Proteins from a sampleare routed over the chip, and the relevant are bound by the ligand.After a washing step, the eluted proteins are analyzed by MALDI-TOF massspectrometry. The system may be a fully automated process and isapplicable to detecting and characterizing proteins present in complexbiological fluids and cell extracts at low- to subfemtomol levels.

Methods of Treatment

The invention farther encompasses a method for improving theeffectiveness of cancer treatment in a subject with cancer. The methodcomprises measuring the level of prohibitin in a biological sample,wherein an elevated level of prohibitin as compared to a standard levelindicates that a subject is resistant to treatment with a member of thetaxoid family and is need of an alternative treatment so as to improvethe effectiveness of cancer treatment

In one embodiment the method comprises serially monitoring a level ofprohibitin in a sample obtained from the subject during a period oftreatment with a taxoid family member; and comparing the level measuredto a standard level, wherein elevation of the measured level of relativeto the standard level indicates that the subject is in need ofadditional treatment.

In one embodiment, a method for treating cancer a is provided whereinthe patient is administered an inhibitor of prohibitin. In oneembodiment, the cancer is a taxoid family member resistant cancer. Thepatient may be administered a taxoid family member compoundconcurrently, prior to, or subsequent to administration of a prohibitininhibitor. In one embodiment, the patient is administered an additionalcompound or agent that reverses taxoid family member resistance (e.g.inhibitor of glutathione-S-transferase π, inhibitor of p-glycoproteinetc.) and/or is administered a compound or agent that has anti-canceractivity.

The inhibitor can prevent the accumulation at any step of the pathwayfrom the gene to protein activity, e.g. preventing transcription,reducing mRNA levels, preventing translation, or inhibiting the proteinitself; e.g. inhibiting translocation of prohibitin to the cell surface,or inhibiting prohibitin at the cell surface. Such inhibitors caninclude antibodies, small molecules (drugs or compounds), antisensemolecules, siRNA, ribozymes, repressors of gene transcription, orcompetitive or non-competitive molecular inhibitors of the gene productart.

In one embodiment, prohibitin inhibitor is administered to subject whodoes not have a taxoid family member resistant cancer. Means fordetermining taxoid family member resistance and sensitivity are wellknown to those skilled in the

In one embodiment, treatment may involve a combination of treatments,including, but not limited to a prohibitin inhibitor in combination withchemotherapy, radiation, or drugs/agents known to be effective againstcancer.

In connection with the administration of a prohibitin inhibitor or otheranti-cancer agent, a drug which is “effective against” a cancerindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as a improvement of symptoms, a cure, a reduction indisease load, reduction in tumor mass or cell numbers, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating theparticular type of disease or condition.

The term “drug” or “compound” or “agent” as used herein refers to achemical entity or biological product, or combination of chemicalentities or biological products, administered to a person to treat orprevent or control a disease or condition. The chemical entity orbiological product is preferably, but not necessarily a low molecularweight compound, but may also be a larger compound, for example, anoligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, sIRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of, adverse physiological effects.

Compounds that specifically target prohibitin, whether detected In vivaor in vitro, can be selected using techniques known in the art anddiscussed herein. Candidate drag screening assays may be used toidentify bioactive candidate agents that inhibit the activity ofprohibitin. Of particular interest are screening assays for agents thathave a low toxicity for human cells. A wide variety of assays may beused for this purpose, including labeled In vitro protein-proteinbinding assays, electrophoretic mobility shift assays, enzyme activityassays, immunoassays for protein binding, and the like. Purifiedprofiling may also be used for determination of three-dimensionalcrystal structure, which can be used for modeling intermolecularinteractions, transporter function, etc. Such compounds may be forexample, small molecules, antibodies, aptamers, siRNAs, and vectors thatinhibit prohibitin activity which confers resistance to a taxoid familymember.

In one embodiment, compounds useful in the method of the presentinvention are antibodies which interfere prohibitin activity, includingmonoclonal, chimeric humanized, and recombinant antibodies and fragmentsthereof which are characterized by their ability to inhibit the taxoidfamily member resistance activity of prohibitin and which have lowtoxicity.

Neutralizing antibodies are readily raised in animals such as rabbits ormice by immunization with prohibitin. Immunized mice are particularlyuseful for providing sources of B cells for the manufacture ofhybridomas, which in turn are cultured to produce large quantities ofanti-prohibitin monoclonal antibodies. Chimeric antibodies areimmunoglobin molecules characterized by two or more segments or portionsderived from different animal species. Generally, the variable region ofthe chimeric antibody is derived from a non-human mammalian antibody,such as murine monoclonal antibody, and the immunoglobin constant regionis derived from a human immunoglobin molecule. Preferably, both regionsand the combination have low immunogenicity as routinely determined.Humanized antibodies are immunoglobin molecules created by geneticengineering techniques in which the murine constant regions are replacedwith human counterparts while retaining the murine antigen bindingregions. The resulting mouse-human chimeric antibody should have reducedimmunogenicity and improved pharmacokinetics in humans.

In one embodiment, an antisense strategy may be used to interfere withthe taxoid resistance activity of prohibitin. This approach may, forinstance, utilize antisense nucleic acids or ribozymes that blocktranslation of a specific mRNA, either by masking that mRNA. with anantisense nucleic acid or cleaving it with a ribozyme. For a generaldiscussion of antisense technology, see, e.g., Antisense DNA and RNA,(Cold Spring Harbor Laboratory, D. Melton, ed., 1988).

Reversible short inhibition of prohibitin gene transcription may also beuseful. Such inhibition can be achieved by use of siRNAs. RNAinterference (RNAi) technology prevents the expression of genes by usingsmall RNA molecules such as small interfering RNAs (siRNAs). Thistechnology in turn takes advantage of the fact that RNAi is a naturalbiological mechanism for silencing genes inmost cells of many livingorganisms, from plants to insects to mammals (McManus at al., NatureReviews Genetics; 2002, 3(10) p. 737). RNAi prevents a gene fromproducing a functional protein by ensuring that the moleculeintermediate, the messenger RNA copy of the gene is destroyed. siRNAscan be used in a naked form and incorporated in a vector, as describedbelow. One can further make use of aptamers to specifically inhibitvariant FTase gene transcription, see, for example, U.S. Pat. No.6,699,843. Aptamers useful in the present invention may be identifiedusing the SELEX process. The methods of SELBX have been described in,for example, U.S. Pat. Nos. 5,707,796, 5,763,177, 6,011,577, 5,580,737,5,567,588, and 5,660,985. The nucleic acid sequence of human Prohibitinis found in Genebank at NM_(—)002634.

The siRNAs may be chemically synthesized, produced using in vitrotranscription, etc. SiRNAs have been described in Brummelkemp et al.,Science 296; 550-555, 2002, Jaque et al., Nature 418; 435-438, 2002,Elbashir S. M. at al. (2001) Nature, 494-498, McCaffrey et al. (2002),Nature, 418: 38-39; Xia H. at al. (2002), Nat. Biotech. 20: 1006-1010,Novina et al. (2002), Nat. Med. 8: 681-686, and U.S. Application No.20030198627.

The delivery of siRNA to tumors can potentially be achieved via any ofseveral gene delivery “vehicles” that are currently available. Theseinclude viral vectors, such as adenovirus, lentivirus, herpes simplexvirus, vaccinia virus, and retrovirus, as well as chemical-mediated genedelivery systems (for example, liposomes), or mechanical DNA deliverysystems (DNA guns). The oligonucleotides to be expressed for suchsiRNA-mediated inhibition of gene expression would be between 18 and 28nucleotides in length.

An “antisense nucleic acid” or “antisense oligonucleotide” is a singlestranded nucleic acid molecule, which, on hybridizing under cytoplasmicconditions with complementary bases in a RNA or DNA molecule, inhibitsthe latter's role. lithe RNA is a messenger RNA transcript, theantisense nucleic acid is a countertranscript or mRNA-interferingcomplementary nucleic acid. As presently used, “antisense” broadlyincludes RNA-RNA interactions, RNA- DNA interactions, ribozymes, RNA),aptamers and Rnase-H mediated arrest. Antisense nucleic acid moleculescan be encoded by a recombinant gene for expression in a cell (e.g.,U.S. Pat. No 5,814,500; U.S. Pat. No. 5,811,234), or alternatively theycan be prepared synthetically (e.g., U.S. Pat. No. 5,780,607).

In one embodiment, the administered inhibitor may be an antisenseoligonucleotides, particularly synthetic oligonucleotides; havingchemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the prohibitin gene, andinhibits expression of the targeted gene products (see e.g. Nyce et al.(1997) Nature 385:720). Antisense molecules inhibit gene expression byreducing the amount of mRNA available for translation, throughactivation of RNAse H or stork hindrance. One or a combination ofantisense molecules may be administered, where a combination maycomprise multiple different sequences from a single targeted gene, orsequences that complement several different genes. Generally, theantisense sequence will have the same species of origin as the animalhost, species variants of prohibitin are available in Genebank e.g.,mouse NM_(—)008831.3, NP_(—)032857; Rat NM_(—)031851.2, NP_(—)114039.Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where the vector isintroduced and expressed in the targeted cells. The transcriptionalinitiation will be oriented such that the antisense strand is producedas an RNA molecule.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these ribozymes,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1989,Science 245(4915) p. 276). Because they are sequence-specific, onlymRNAs with particular sequences are inactivated.

Aptamers are also useful. Aptamers are a promising new class oftherapeutic oligonucleotides or peptides and are selected in vitro tospecifically bind to a given target with high affinity, such as forexample ligand receptors. Their binding characteristics are likely areflection of the ability of oligonucleotides to form three dimensionalstructures held together by intramolecular nucleobase pairing. Aptamersare synthetic DNA, RNA or peptide sequences which may be normal andmodified (e.g. peptide nucleic acid (PNA), thiophophorylated DNA, etc)that interact with a target protein, ligand (lipid, carbohydrate,metabolite, etc). In a further embodiment, RNA aptamers specific forprohibitin can be introduced into or expressed in a cell as atherapeutic.

Peptide nucleic acids (PNAs) are compounds that in certain respects aresimilar to oligonucleotides and their analogs and thus may mimic DNA andRNA. PNA'S are suitable inhibitors for use in embodiments of theinvention. In PNA, the deoxyribose backbone of oligonucleotides has beenreplaced by a pseudo-peptide backbone (Nielsen et al. 1991 Science 254,1457-1560). Each subunit, or monomer, has a naturally occurring ornon-naturally occurring nucleobase attached to this backbone. One suchbackbone is constructed of repeating units of N-(2-aminoethyl) glycinelinked through amide bonds. PNA hybridises with complementary nucleicacids through Watson and Crick base pairing and helix formation. ThePseudo-peptide backbone provides superior hybridization properties(Egholm et al. Nature (1993) 365, 566-568), resistance to enzymaticdegradation (Demidov et al. Biochem. Pharmacol. (1994) 48, 1310-1313)and access to a variety of chemical modifications (Nielsen and HaaimaChemical Society Reviews (1997) 73-78). PNAs specific for a variantFTase can be introduced into or expressed in a cell as a therapeutic.PNAs have been described, for example, in U.S. Application No.20040063906.

Once identified, such inhibitor compounds are administered to patientsin need of taxoid resistance treatment, for example, patients affectedwith or at risk for developing taxoid resistance.

The route of administration of inhibitors or other therapeuticcompounds/complexes of the invention may be intravenous (I.V.),intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.),intraperitoneal (I.P.), intrathecal (I.T.), intrapleural, intrauterine,rectal, vaginal, topical, intratumor and the like. The therapeuticcompounds/compositions of the invention can be administered parenterallyby injection or by gradual infusion over time and can be delivered byperistaltic means.

Administration may be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the compounds of the inventionare formulated into conventional oral administration forms such ascapsules, tablets and tonics.

For topical administration, the pharmaceutical composition (inhibitor ofkinase activity) is formulated into ointments, salves, gels, or creams,as is generally known in the art.

The therapeutic compositions of this invention are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual.

Any formulation or drug delivery system containing the therapeutic,which is suitable for the intended use, as are generally known to thoseof skill in the art, can be used. Suitable pharmaceutically acceptablecarriers for oral, rectal, topical or parenteral (including inhaled,subcutaneous, intraperitoneal, intramuscular and intravenous)administration are known to those of skill in the art. The carrier mustbe pharmaceutically acceptable in the sense of being compatible with theother Ingredients of the formulation and not deleterious to therecipient thereof.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials am capable ofadministration to or upon a mammal without the production of undesirablephysiological effects.

Patients to be treated with a compound which inhibits or targetsprohibitin include, for example, patients diagnosed with taxoid familymember resistance, patients who initially respond to therapy with ataxoid family member, but subsequently fail to respond to the same orsimilar compound. Compounds can be combined that target multipleproteins involved in taxoid family member resistance.

Another embodiment of the invention provides methods of treatingsubjects with tumid family member resistant cancers. In particular,taxoid family member resistant cancers with elevated levels ofprohibitin on the cell surface of cancer cells. The method comprisesadministering a compound that comprises and agent that selectively bindsprohibitin and a therapeutic agent.

In one embodiment, the agent that selectively binds prohibitin is apeptide.

As used herein, a “protein” or “peptide” generally refers, but is notlimited to, a protein of greater than about 200 amino acids up to a fulllength sequence translated from a gene; a polypeptide of about 100 to200 amino acids; and/or a “peptide” of from about 3 to about 100 aminoacids. For convenience, the terms “protein,” “polypeptide” and “peptide”are used interchangeably herein,

As used herein, an “amino acid” refers to any naturally occurring aminoacid, any amino acid derivative or any amino acid mimic known in theart. In certain embodiments, the residues of the protein or peptide aresequential, without any non-amino acid interrupting the sequence ofamino acid residues. in other embodiments, the sequence may comprise oneor more non-amino acid moieties. In particular embodiments, the sequenceof residues of the protein or peptide may be interrupted by one or morenon-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally warring proteins, or at least one modified or unusual aminoacid.

The agent that selectively binds prohibitin (e.g. the targeting proteinor peptide) can be attached to a therapeutic agent to form a complex.For example, this can be done by the generation of fusion proteins. Suchmolecules generally have all or a substantial portion of the targetingpeptide (i.e. the protein or peptide that selectively binds prohibitin),linked at the N- or C-terminus, to a portion of a second polypeptide orprotein (i.e. therapeutic agent). Examples of proteins or peptides thatmay be incorporated into a fusion protein with the peptide or proteinthat selectively binds prohibitin include cytostatic proteins, cytocidalproteins, pro-apoptosis agents, anti-angiogenic agents, hormones,cytokines, growth factors, peptide drugs, antibodies, Fab fragmentsantibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins,cell adhesion proteins and binding proteins. These examples are notmeant to be limiting and it is contemplated that within the scope of thepresent invention virtually any protein or peptide could be incorporatedinto a fusion protein comprising a targeting peptide that selectivelybinds prohibitin. Methods of generating fusion proteins are well knownto those of skill in the art. Such proteins can be produced, forexample, by chemical attachment using bifunctional cross-linkingreagents, by de novo synthesis of the complete fusion protein, or byattachment of a DNA sequence encoding the targeting peptide to a DNAsequence encoding the second peptide or protein, followed by expressionof the intact fusion protein.

Alternatively, the agent that selectively bind prohibitin may beattached to therapeutic agents using a variety of known cross-linkingagents. Methods for covalent or non-covalent attachment of proteins orpeptides are well known in the art. Such methods may include, but arenot limited to, use of chemical cross-linkers, photoactivatedcross-linkers and/or bifunctional cross-linking reagents. Exemplarymethods for cross-linking molecules are disclosed in U.S. Pat. No.5,603,872 and U.S. Pat. No. 5,401,511, incorporated herein by reference.Non-limiting examples of cross-linking reagents of potential use includeglutaraldehyde, bifunctional oaken; ethylene glycol diglycidyl ether,carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ordicyclohexylcarbodiimide, bisimidates, dinitrobenzene,N-hydroxysuccinimide ester of =bale acid, disuccinimidyl tartarate,dimethyl-3,3′-dithio-bispropionimidate, azidoglyoxal,N-succinimidyl-3-(2-pyridyldithio)propionate and4-(bromoadminoethyl)-2-nitrophenylazide.

Homobifunctional reagents that carry two identical functional groups arehighly efficient in inducing cross-linking. Heterobifunctional reagentscontain two different functional groups. By taking advantage of thedifferential reactivities of the two different functional groups,cross-linking can be controlled both selectively and sequentially. Thebifunctional cross-linking reagents can be divided according to thespecificity of their functional groups, e.g., amino, sulfhydryl,guanidino, indole, carboxyl specific groups. Of these, reagents directedto free amino groups have become especially popular because of theircommercial availability, ease of synthesis and the mild reactionconditions under which they can be applied.

In certain embodiments, it may be appropriate to link one or more agentsto a liposome or other membrane-bounded particle. For example, targetingpeptides or agents and therapeutic agents can be cross-linked toliposomes, microspheres or other such devices may be used to deliverlarger volumes of a therapeutic agent to a target organ, tissue or celltype. Various ligands can be covalently bound to liposomal surfacesthrough the cross-linking of amine residues. Liposomes containingphosphatidylethanolamine (PE) may be prepared by established procedures.The inclusion of PE provides an active functional amine residue on theliposomal surface. In another non-limiting example, heterofunctionalcross-linking reagents and methods of use are disclosed in U.S. Pat. No.5,889,155, incorporated herein by reference. The cross-linking reagentscombine a nucleophilic hydrazide residue with an electrophilic maleimideresidue, allowing coupling in one example, of aldehydes to free thiols.The cross-linking reagent can be modified to cross-link variousfunctional groups.

In one embodiment, the peptide that selectively binds prohibitin isCKGGEAKDC SEQ ID NO: 2 (See U.S. Patent Application Publication2006/0094672).

In one embodiment, the protein or peptide that selectively bindsprohibitin is an antibody-based binding moiety.

Other techniques of general use for proteins or peptides that are knownin the art have not been specifically disclosed herein, but may be usedin the practice of the claimed subject matter.

Therapeutic Agents

Therapeutic agents or factors suitable for attaching to an agent thatselectively binds prohibitin may include any chemical compound thatinduces apoptosis, cell death, cell stasis and/or anti-angiogenesis orotherwise affects the survival and/or growth rate of a cancer cell.Examples of some of these compounds are listed below.

Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr at al., 1972). The Bel-2 family ofproteins and ICE-Like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role hi controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Tsujimoto et al., 1985). The evolutionarily conserved Bcl-2protein now is recognized to be a member of a family of relatedproteins, which can be categorized as death agonists or deathantagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bel-2 cell death regulatory proteins thatshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S, Mcl-1, A1, Bfl-1) orcounteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik,Bim, Bid, Bad, Harakiri).

Non-limiting examples of pro-apoptosis agents contemplated within thescope of the present invention include gramicidin, magainin, mellitin,defensin, cecropin, (KLAKLAK)₂(SEQ ID NO: 3), KLAKKLA)₂(SEQ ID NO:4),(KAAKKAA)₂ (SEQ ID NO:5) or (KLGKKLG)₃(SEQ ID NO:6).

Angiogenic Inhibitors

In certain embodiments the present invention may concern administrationof targeting peptides attached to anti-angiogenic agents, such asangiotensin laminin peptides, fibronectin peptides, plasminogenactivator inhibitors, tissue metalloproteinase inhibitors, interferons,interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

Proliferation of tumors cells relies heavily on extensive tumorvascularization, which accompanies cancer progression. Thus, inhibitionof new blood vessel formation with anti-angiogenic agents and targeteddestruction of existing blood vessels have been introduced as aneffective and relatively non-toxic approach to tumor treatment. (Arap etal., Science 279:377-380,1998a; Arap et al., Curr. Opin. Oncol.10:560-565, 1998b; Ellerby et al., Nature Med. 5:1032-1038, 1999). Avariety of anti-angiogenic agents and/or blood vessel inhibitors areknown. (E.g., Folkman, In: Cancer: Principles and Practice, eds. DeVitaet al., pp. 3075-3085, Lippincott-Raven, New York, 1997; Eliceiri andCheresh, Curr. Opin. Cell. Biol. 13, 563-568, 2001).

Cytotoxic Agents

A wide variety of anticancer agents are well known in the art and anysuch agent may be coupled to a cancer targeting peptide for use withinthe scope of the present invention, Exemplary cancer chemotherapeutic(cytotoxic) agents of potential use include, but are not limited to,5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide(VP16), farnesyl-protein transferase inhibitors, gemcitabine,ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine,nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol,temazolomide (an aqueous form of (DTIC), transplatinum, vinblastine andmethotrexate, vincristine, or any analog or derivative variant of theforegoing. Most chemotherapeutic agents fall into the categories ofalkylating agents, antimetabolites, antitumor antibiotics,corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormoneagents, miscellaneous agents, and any analog or derivative variantthereof.

Chemotherapeutic agents and methods of administration, dosages, etc. arewell known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and “Remington: The Science and Practice ofPharmacy,” 20th edition, Gennaro, Lippincott, 2000, each incorporatedherein by reference in relevant parts), and may be combined with theinvention in light of the disclosures herein. Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Of course,all of these dosages and agents described herein are exemplary ratherthan limiting, and other doses or agents may be used by a skilledartisan for a specific patient or application. Any dosage in-betweenthese points, or range derivable therein is also expected to be of usein the invention.

Alkylating Agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent cells from proliferating. This category of chemotherapeuticdrugs represents agents that affect all phases of the cell cycle, thatis, they are not phase-specific. An alkylating agent, may include, butis not limited to, nitrogen mustard, ethylenimene, methylmelamine, alkylsulfonate, nitrosourea or triazines. They include but are not limitedto: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.

Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,they specifically influence the cell cycle during S phase.Antimetabolites can be differentiated into various categories, such asfolic acid analogs, pyrimidine analogs and purine analogs and relatedinhibitory compounds. Antimetabolites include but are not limited to,5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, andmethotrexate.

Natural Products

Natural products generally refer to compounds originally isolated from anatural source, and identified as having a pharmacological activity.Such compounds, analogs and derivatives thereof may be, isolated from anatural source, chemically synthesized or recombinantly produced by anytechnique known to those of skill in the art. Natural products includesuch categories as mitotic inhibitors, antitumor antibiotics, enzymesand biological response modifiers.

Antibiotics

Certain. antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Examples ofcytotoxic antibiotics include, but are not limited to, bleomycin,dactinomycin, daunorubicin, doxorubicin (Adriamycin); plicamycin(mithramycin) and idarubicin.

Miscellaneous Agents

Miscellaneous cytotoxic agents that do not fall into the previouscategories include, but are not limited to platinum coordinationcomplexes, anthracenediones, substituted ureas, methyl hydrazinederivatives, amsacrine, L-asparaginase, and tretinoin. Platinumcoordination complexes include such compounds as carboplatin andcisplatin (cis-DDP). An exemplary anthracenedione is mitoxantrone. Anexemplary substituted urea is hydroxyurea. An exemplary methyl hydrazinederivative is procarbazine (N-methylhydrazine, MIH). These examples arenot limiting and it is contemplated that any known cytotoxic, cytostaticor cytocidal agent may be attached to targeting peptides andadministered to a targeted organ, tissue or cell type within the scopeof the invention.

Cytokines and Chemokines

In certain embodiments, it may be desirable to couple specific bioactiveagents to one a targeting peptide that selectively binds prohibitin.Such agents include, but are not limited to, cytokines and/orchemokines.

The term “cytokine” is a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof cytokines are lymphokines, monokines, growth factors and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-β; platelet growth factor, transforming growthfactors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-.alpha., -.beta, and -.gamma.; colony stimulating factors(CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1,IL-1.alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIP, G-CSF, GM-CSF,M-CSF, EPO, kit-ligand or FLT-3, anglostatin, thrombospondin,endostatin, tumor necrosis factor and LT. As used herein, the termcytokine includes proteins from natural sources or torn recombinant cellculture and biologically active equivalents of the native sequencecytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to the site of treatment. Chemokines include, but are notlimited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the terrachemokines.

Dosages

The skilled artisan is directed to “Remington: The Science and Practiceof Pharmacy,” 20th edition, Gennaro, lippincott (2000). Some variationin dosage will. necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by theFDA Office of Biologics standards.

Kits

In another aspect, the invention concerns an article of manufacture orpackage, comprising a container, a composition within the containercomprising a prohibitin antagonist (inhibitor of prohibitin), e.g., ananti-prohibitin antibody, optionally a label on or associated with thecontainer that indicates that the composition can be used for treating acondition characterized by resistance to a taxoid family member, and apackage insert containing instructions to administer the antagonist topatients who have been found to have taxoid family member resistance,e.g. elevated levels of prohibitin. A therapeutic product may includesterile saline or another pharmaceutically acceptable emulsion andsuspension base.

In another embodiment, the invention provides s an article ofmanufacture or package, comprising a container, a composition within thecontainer comprising an agent (e.g. protein or peptide) that selectivelybinds prohibitin, complexed to a therapeutic agent, optionally a labelon or associated with the container that indicates that the compositioncan be used for treating cancer.

Embodiments of the invention are also directed to commercial kits forthe detection of levels of prohibitin. The kit can be in anyconfiguration well known to those of ordinary skill in the art and isuseful for performing one or more of the methods described herein forthe detection of prohibitin. The kits are convenient in that they supplymany if not all of the essential reagents for conducting an assay orassays for the detection of prohibitin. In addition, the assay ispreferably performed simultaneously with a standard or multiplestandards that are included in the kit, such as a predetermined amountof protein so that the results of the test can be quantitated orvalidated.

The kits include a means for detecting prohibitin protein, such asantibodies, or antibody fragments, which selectively bind to prohibitin.The detection kit can be formulated in a standard two-antibody bindingformat in which, for example, one specific antibody captures prohibitinin a patient sample and another specific antibody is used to detectcaptured protein. For example, the capture antibody is immobilized on asolid phase, e.g., an assay plate, an assay well, a nitrocellulosemembrane, a bead, a dipstick, or a component of an elution column. Thesecond antibody, i.e., the detection antibody, is typically tagged witha detectable label such as a calorimetric agent or radioisotope.

In other embodiments, the detection kits may employ, but are not limitedto, the following techniques: competitive and non-competitive assays,radioimmunoassay (RIA) bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELBA, microliter plates, andimmunocytochemistry or immunohistochemistry. For each kit the range,sensitivity, precision, reliability, specificity and reproducibility ofthe assay are established by means well known to those skilled in theart.

In one embodiment, the detection kit may include means for the detectionof other biomarkers, e.g., other cancer markers, e.g., other drugresistance markers.

The above described detection kits would further provide instructionsfor use.

EXAMPLES Materials and Methods

Materials. Paclitaxel was purchased from Sigma-Aldrich, St. Louis, Mo.The drug was prepared as a 5 mM stock in ethanol and stored in aliquotsat −20° C. Working stock solutions were diluted in DMSO and furtherdiluted in culture medium at appropriate concentrations. The IPG strips,criterion gels, ampholytes and Syproruby stain were purchased fromBiorad (city). Phenanthroline, Benzamidine, and PMSF were purchased fromSigma-Aldrich. The following antibodies were used: rabbit anti humanprohibitin (BioLegend, city Calif.) mouse anti-human GST-π (BioLegend),GAPDH (Abcam, Cambridge, Mass.), anti-mouse actin (Abcam, Cambridge,Mass.) Alexa Fluor 368 goat anti-mouse IgG and Alexa Fluor 488anti-rabbit IgG (Molecular Probes, Eugene, Ore.) mouse anti-humanVDAC/Porin (Abcam, Cambridge, Mass.). SiRNA oligonucleotides werepurchased as SMARTpool reagents (Dharmacon, Lafayette, Colo.).

Cell Culture. The human non-small cell lung carcinoma cell line A549 andits Paclitaxel-resistant derivative cell lines were cultured in F-12 Hamnutrient mixture (Invitrogren, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (FBS) at 37° C. in a humidified atmosphere with 5%CO₂/95% air. The pacliturel-resistant cell lines were grown underselective pressure (100 nM paclitaxel), then placed in Paclitaxel-freeculture medium 5-7 days before experiments were performed. The uterinesarcoma cells (MES-SA) and the multidrug resistant derivative(MES-SA/DX5) were maintained in McCoy's 5A media (ATCC, Rockville, Md.)supplemented with 10% FBS. The drug-resistant MES-SA/DX5 cells(originally selected with doxorubicin) were initially adapted topaclitaxel by stepwise increase of concentration from 5 nM to 100 nMover a 60-day period. They were subsequently routinely grown in 100 nMpaclitaxel, which was withdrawn 5-7 days before experiments wereperformed.

Protein fractionation. To prepare protein fractions for two-dimensionalelectrophoresis, cells from the parental cell lines and its drugresistant cell lines were grown in five 150 mm tissue culture disheseach. When the cells reached confluency (>80%) they were removed byscrapping into Ca⁺⁺/Mg⁺⁺ free PBS, and washed twice with PBS containingprotease inhibitors (5 mM phenanthroline, 5 mM benzamidine and 1 mMPMSF). The cells were then resuspended in a hypotonic solution of 50%PBS homogenized in a glass homogenizer with 20 gentle strokes andcentrifuged at 355×g for 10 mins. Nuclear proteins were recovered fromthe pellet by low-speed centrifugation. The supernatant was centrifugedseparately at 27,000×g for 45 minutes to separate the microsomes(pellet) from the cytoplasmic components. The nuclear and microsomalpellets were resuspended in 10 mM Tris-HCl huffier containing 150 mMNaCl, 1% Triton X-114 and protease inhibitors (which ones, whatconcentrations) and incubated on ice for 10 min. The three proteinfractions were diluted separately in 10 mM NH⁴HCO₃ containing 2% CHAPSand concentrated using a Centricon-20 spin column (Millipore, Billerica,Mass.). The protein concentrates were lyophilized and stored at 20° C.prior to two-dimensional electrophoresis. Separate aliquots (10 μl) wereprepared for protein quantification.

Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE). Thelyophilized protein fractions were dissolved in 2D-PAGE buffer (8M urea,2% CHAPS, 100 mM DTT and 0.2% ampholytes) at room temperature. Thedissolved proteins were rehydrated on dry IPG strips (pH gradient of 4-7Biorad) for 16 h-24 h at room temperature. The proteins (50 μg) wereanalyzed by two-dimensional electrophoresis consisting of anisoelectric-focusing (IEF) step followed by electrophoretic separationusing a 4-20% gradient criterion gel (Biorad). Gels were stained withSyproRuby fluorescence stain according to the manufacturer's (Biorad)protocol. All samples were run at least twice to ensure reproducibility.Image analysis was performed on a scanner using PDQuest software fromBiorad. Only spots that were differentially expressed in the resistantsublines of both lung cancer and uterine sarcoma cell lines wereselected for detailed investigation.

In-Gel Trypsin Digestion and Protein Identification by Tandem MassSpectrometry

Spots of interest were excised with a PDM1.5 manual spot picker (The GelCompany, San Francisco Calif.). The gel plugs were washed in 100 mMammonium bicarbonate (NH₄HCO₃) solution for 1 h and then incubated with0.5 ml 1:1 mixture of 50 mM NH₄HCO₃ and 50% acetonitrile for 30 minutes.Complete dehydration was achieved by incubating the gel plugs with 200μl of 100% acetonitrile and the gel pieces were dried in a SpeedVac for20 minutes. For in gel digestion the dried gel particles were rehydratedwith a minimal volume of trypsin solution (10 μg/μl in 25 mM NH₄HCO₃)and incubated at 37° C. overnight. The digests were extracted with 50%acetonitrile twice and the resulting pooled protein concentrated with aSpeedVac to approximately 10% of the volume. Protein identification wasperformed using an Ion-Trap tandem mass spectrometer (Maker, CityState). Samples were analyzed by nanoflow HPLC micro-electrosprayionization on a Finnigan LCQ ion trap spectrometer (Thermo-Finnigan SanJose, Calif.). A gradient from 95% A to 80% A for 30 min then to 75% Afor 15 min and finally to 10% A for 60 min (where A=0.1% formic Acid inwater, B=0.1% formic acid in acetonitrile, (Burdick and Jackson)) wasrun at 200 ml/min over a self packed, flame pulled C-18 integratedcolumn and electrospray emitter into the LCQ Deca. The 100 μm ID×8 cmfused silica column (Polymicro Technologies, Phoenix Ariz.) was slurrypacked with Magic C18AQ 200 Å, (Michrom BioResources Auburn, Calif.).Spectra were acquired in a data dependent mode throughout the gradient,a full MS scan followed by 3 subsequent ms/ms scans based of the 3 mostintense peaks in the previous full scan. CID fragmentation was achievedby collision energy of 35%, the capillary was heated to 150° C. andelectro spray voltage was 1.9 KV. Once an ms/ms spectra was obtained twotimes it was put on an exclusion list for 3 min to allow for lowerintensity peptides to be analyzed. Data was analyzed using the Sequestalgorithm by searching against the updated non-redundant database fromNCBI. Spectra are ranked by X-correlation score.

Western Blots. Proteins in membrane fractions, cytoplasmic fractions orwhole cell lysates were resolved by SDS-PAGE electrophoresis andtransferred to PVDF membranes. The membranes were blotted for moleculesof interest with anti-prohibitin (1:1000), anti-GST-π (1:1000),anti-GAPDH (1:1000) and anti-actin (1:1000) antibodies for at roomtemperature for 1 hour or overnight at 4° C. The bound primaryantibodies were detected using appropriate horseradishperoxidase-conjugated secondary antibodies followed by detection withenhanced chemiluminescense (Perkin Elmer, Wellesley, Mass.). Forsuccessive blotting, the membranes were treated with stripping buffer(Chemicon, Calif.) for 15 minutes and than re-analyzed using appropriateantibodies.

Cytotoxicity Assays. Cell growth inhibition was determined by3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide assayusing MTT (Sigma Chemicals, St. Louis, Mo.) reagent in 96-well plates.Briefly, approximately 1×10⁴ cells were plated in 96-well plate and 24hours later paclitaxel was added in appropriate concentrations. After 72hours of drug incubation, 50 μl of MTT reagent was added to each welland incubated for an additional 4 hours. 200 μl of isopropanol-HClsolution was added to each well to dissolve the cell pellets. Absorbancewas determined using a SpectraMax plate reader (Molecular Devices,Sunnyvale, Calif.) at 570 nm.

In vitro Transfection with Si RNA: The human prohibitin mRNA-specificRNA oligonucleotides and human GST-π mRNA-specific oligonucleotides werepurchased as SmartPool mixtures (combinations of four differentoligonucleotides) from Dharmacon (Lafayette, Colo.). A non-specificscrambled RNAi duplex was used as a negative control. 2.5×104 cells wereseeded in 24 well dishes 1 day prior to transfection. Cells weretransfected with the appropriate siRNA's using SilentFect (Biorad)reagent according to manufacturers protocol (confluency ˜50%-70% at thetime of transfection). The transfection mixture was prepared by mixing625 μl of serum-free medium containing 8 μl of 20 μM siRNA with 625 μlof serum-free media containing 18 μl of SilentFect reagent. Beforetransfection, the medium in the 24 wells was replaced with 250 μl offresh medium. The control siRNA mixture was prepared by mixing 625 μl ofserum-free medium containing 3 μl of 50 μM control siRNA with 625 μlserum-free mediate containing 18 μl of SilentFect reagent Thetransfection mixture (50 μl) was added to the 24-well plate within 20min after mixture preparation in a total volume of 300 μl (finalconcentration of 20 nM siRNA). The cells were incubated for 4 h at 37°C., after which the medium in the 24-well plate was replaced with 300 μlof fresh medium. Cells were treated with control medium or withpaclitaxel at 10 nM or 100 nM 24 h after transfection with specificsiRNA or control siRNA as described above. Cells were counted for cellnumber and harvested for western blotting 72 h after paclitaxeltreatment. Effect of paclitaxel on siRNA treated cells was computed as %cell survival.

Immunofluorescence and Confocal Microscopy

Cells were seeded on round glass coverslips coated with 10 μg/mlfibronection (BD Biosciences) in 24 well plates. Cells were stained formitochondria either with 400 nM Mitotracker Red, a live-cell stain formitochondria or with antibodies to VDAC (Abcam, Cambridge, Mass.) amitochondrial porin channel protein located on the outer membrane of themitochondria. Cells were incubated for 45 min at 37° C. in mediumcontaining 400 nM of Mitotracker. The medium was removed, and the cellswashed with prewarmed media. Cells were fixed with 4% paraformaldehydefor 10 min at room temperature and permeabilized with ice-cold acetonefor 5 min at room temperature. Cells were then washed with PBS andblocked for 30 min in PBS containing 1% BSA. Cells were then stained forprohibitin by first incubating them in PBS containing 1% BSA and rabbitanti-prohibitin (1:100) for 1 h at room temperature followed by Alexagreen 488 conjugated anti-rabbit IgG for 30 min in dark. The cells werethen rinsed with PBS, followed by distilled water. The resultingcoverslips were mounted on glass slides with Vectashield Mounting mediacontaining DAPI (Burlingame, Calif.). The cells were analyzed using aLeica (Bannockburn, Ill.) DMIRE2 SP2 confocal microscope equipped withan Acousto-Optical Beam Splitter (AOBS) and X 40 oil immersion objectiveoperated with differential interference contrast (DIC) and fluorescence.Fluorescence was excited sequentially; first by a 488-nm line of anargon-krypton laser with a recorded emission from 500 to 572 nm, andthen by a 543-nm line of a Helium Neon Laser recorded emission from 613to 700 nm.

Results

Protein Profiling by Two-Dimensional Gel Electrophoresis and MassSpectrometry Identifies Proteins Associated with Paclitaxel Resistance

We adopted a proteomics based approach using two-dimensional gelelectrophoresis coupled with mass spectrometry to identify novelproteins associated with paclitaxel resistance. Two different cancercell lines were used in the study; 1) a paclitaxel-sensitive lung cancercell line (A549) and its paclitaxel-resistant variant (A549-TR)(ChouCancer Res 2005), 2) a uterine sarcoma cell line (MES-SA) and itspaclitaxel-resistant variant (MES-SA/DX5). The resistant sublimesdiffered from the parental lines in their sensitivity to paclitaxel bygreater than 100-fold (FIGS. 1A and B). Cytoplasmic and, microsomalfractions from the parental and paclitaxel-resistant salines wereprepared, as described in materials and methods and separated bytwo-dimensional gel electrophoresis. The purpose of this fractionationstep was to enable identification of low-abundant proteins that wouldotherwise be undetected in total protein lysates as well as to detectpossible translocations of proteins from one site to another. Analysisof the gels revealed more than 50 proteins differentially expressedbetween A549 (parental) and A549-TR (paclitaxel-resistant) cell lines.Examination of the respective fractions from the MES-SA (parental) andMES-SA-DX5 (paclitaxel-resistant) cells also revealed multipledifferentially expressed proteins.

Importantly, we observed a limited number of proteins that weredifferentially expressed in both paclitaxel-resistant cell models.Comparison of A549 and A549TR, MES-SA and MES-SA DX5 two-dimensionalgels of cytoplasmic fractions using computer imaging software (PDQuest)analysis revealed the same protein is overexpressed in the Paclitaxelresistant sublines of both cell types compared to their parental cells(data not shown). Tandem-mass spectrometry was used to identify theprotein as glutathione-S-tranferase-Pi GST-π. Several other proteinsincluding Tubulin β-5, and annexin 1, were found to be overexpressed inthe cytoplasmic fraction of the resistant cell lines, while lipocortinwas relatively underrepresented in this fraction (TABLE 2).

Comparison of the A549 and A549TR, MES-SA and MES-SA DX5 two-dimensionalgels using computer software (PDQuest) analysis revealed the sameprotein is overexpressed in the paclitaxel resistant sublines of bothcell types compared to their parental lines. Tandem-mass spectrometrywas used to identify the protein as Prohibitin (data not shown).Prohibitin, a protein not previously associated with taxane resistance,was consistently overexpressed in the microsomal fractions of theresistant cell lines. GST-π, representative of a cytoplasmic fractionprotein, and prohibitin, representative of a microsomal fractionprotein, were chosen for further investigation of their role inpaclitaxel-resistance.

Validation of Prohibitin and GST-π Protein Expression

GST-π and prohibitin protein levels were examined in whole cell lysatesand cellular fractions by Western blot analysis. Increased levels ofGST-π were seen in the cytoplasmic fractions (FIG. 2A) and in whole celllysates (FIG. 2C) of both resistant cell lines. Although increasedlevels of prohibitin were seen in microsomal fractions (FIG. 213) ofresistant cells, confirming the two-dimensional gel analysis, there wasno difference in the total amount of prohibitin protein seen in wholecell lysates (FIG. 2D). This is an intriguing result as prohibitin hasbeen shown to migrate between intracellular locales, thus theintracellular distribution of prohibitin is relevant to the resistantphenotype.

Cellular Localization of Prohibitin

The potential differential localization of prohibitin inpaclitaxel-resistant cell lines was father investigated using confocalmicroscopy. In both parental (sensitive) minims, A549 and MES-SA,prohibitin staining was predominantly perinuclear (data not shown), andcolocalization determined, Interestingly, the paclitaxel-resistant cells(A549-TR and MES-SA-DX), displayed reduced staining with MitoTracker Redrelative to parental cells and both MitoTracker Red and prohibitinstaining was distributed throughout the cell rather than localized tothe perinuclear region with fewer overlapping regions of co-localization(data not shown). Similar results were obtained using a polyclonalantibody against prohibitin and a monoclonal antibody against themitochondrial protein VDAC/Porin (data not shown). Prohibitin stainingco-localized with the live mitochondrial stain MitoTracker Red in theparental cells (data not shown), suggesting a mitochondrial localizationfor prohibitin in the paclitaxel sensitive cell lines which was lessapparent in the resistant cells (data not shown). Thuspaclitaxel-resistance in these cells is associated with prohibitinrelocalization.

We farther investigated whether differences in cell surface prohibitinwere apparent on placlitaxel-resistant cells. Prohibitin immunostainingwas performed on non-permeabilized cells. Relative to control A549cells, increased surface staining was observed on thepaclitaxel-resistant A549TR (data not shown). Accordingly, cell surfaceprohibitin is an important therapeutic target for the development of newdrugs for the treatment of taxane-resistant tumors.

Effect of Silencing GST-π and Prohibitin on Paclitaxel Sensitivity

To determine the functional significance of the protein changes thatwere correlated with paclitaxel-resistance, we employed SiRNA (DharmaconSmart Pools) to selectively reduce the amounts of these proteins inresistant cell lines. Conditions were chosen in which the protein levelswere reduced by 50-70% for GST-π and prohibitin (FIGS. 3A and 3B insertrespectively). Scrambled, non-specific siRNA, bad no effect on proteinlevels. Knockdown of either GST-π (FIG. 3A) or prohibitin (FIG. 3B)expression in the A549TR cells decreased cell survival by 24% and 20%respectively when the cells were challenged with 10 nM paclitaxel.Similar results were obtained using a single prohibitin siRNA (21 mer)oligonucleotide (Dharmacon) suggesting that the increase in paclitaxelsensitivity is not due to possible off-target effects (data not shown).Intriguingly, simultaneous transfection with GST-π siRNA and prohibitinsiRNA smartpools in the A549TR (FIG. 4A) and MES-SA-DX5 cells (FIG. 4B)dramatically decreased cell survival after exposure to 10 nM paclitaxelrelative to control siRNA. Again similar results were obtained using thesingle prohibitin siRNA (21 mer) oligonucleotide (Dharmacon) (data notshown). These results suggest that combined suppresion of prohibitinalong with GST-π can effectively reverse paclitaxel resistance andrender resistant cells sensitive to the drug.

In this study we used a proteomics approach to identify proteins whoselevel of expression or cellular location are associated with. resistanceto the antitumor drug paclitaxel. The detection of changes in βV-tubulinand GST-π serve as a “proof of principle” for our proteomics basedapproach, as both proteins have previously been implicated inpaclitaxel-resistance in several different studies. Paclitaxel binds tothe β-subunit of tubulin, of which at least seven isotypes exist at theprotein level in humans. These can be classified into two distinctgroups; (1) βI-, βII-, βIVa- and βIVb- tubulin and (2) βIII-, βV-, andβVI-tubulin. Tumor cells often express a different complement ofβ-tubulin isotypes than their normal counterparts. Overexpression,mutation, and post-translational modifications of β-tubulin have allbeen implicated in paclitaxel resistance in cell lines and tumors.Kavallaris et. al. (Br J Ca 1999) showed that paclitaxel-resistant A549cells overexpressed βIII-tubulin and that partial sensitivity topaclitaxel was regained by down-regulation of βIII-tubulin in thesecells. More recently it has been shown that overexpression ofβIII-tubulin induces paclitaxel resistance by reducing the ability ofpaclitaxel to suppress microtubule dynamics (Kamath J B C 2005).Bhattacharya and Cabral showed that mouse 13V tubulin overexpression inCHO cells results in profound microtubule disorganization and dependenceof cells on paclitaxel for growth (14) and more recently Verdier-Pinardet. al. were able to detect βV-tubulin protein in human cell lines andfound that it is highly expressed in Hey, an epithelial ovarian cancercell line.(Pascal Verdier-Pinard, Biochem 2005). Except for theirC-termini, βIII- and, βV-tubulin sequences are closely related. It isnot surprising therefore that we identifiedβV-tubulin as one of theproteins overexpressed in the paclitaxel-resistant cell lines.

Increased levels of GST-β have also been associated with taxaneresistance. GST-π is a detoxification enzyme frequently upregulated intumors and its expression correlates with anticancer drug resistance,especially resistance to alkylating agents such as cisplatin anddoxorubicin. GST-π has also been shown to be a prognostic indicator ofdrug response and survival in non-small cell lung cancer and breastcancer. Clinical studies using TLK286, a GST-π-activated glutathioneanalog prodrug, in platinum and paclitaxel refractory or resistantovarian cancer are currently in progress. (Kavanagh J J et. al. Int JGynecol Cancer. 2005). Our experiments now show that silencing GST-βpartially restores paclitaxel sensitivity in two cellular models of drugresistance. These results provide a direct demonstration of the utilityof GST-πsuppression in sensitizing resistant cells to the effects ofpaclitaxel.

We also present the novel finding of cell surface expression ofprohibitin in paclitaxel-resistant cells. At least two related proteins,prohibit; (Phb-1, BAP32) and Phb2 (BAP37, prohibitone, REA) exist ineukaryotic cells. Prohibitins are well characterized as mitochondrialchaperone proteins that function in a high-molecular-weight complex inthe inner membrane to maintain mitochondria' integrity. However, theirrole outside the mitochondria remains controversial. Mammalianprohibitins have been detected in the cytosol and the nucleus andpostulated to play a role in tumor suppression, inhibition ofproliferation, and apoptosis. Prohibitins have also recently been foundin the plasma membrane of intestinal epithelial cells (Sharma PNAS 2004)and the vasculature of white adipose tissue (Kolonin M G et al 2004 NatMed.). As we also find prohibitin on the surface of paclitaxel-resistantcells, prohibitin can function as a surface-binding site that will be auseful target for therapeutic compounds. Our results further indicatethat the cellular location of prohibitin is altered inpaclitaxel-resistant cells relative to their drug-sensitive parent celllines even though the total levels of prohibitin are not altered in theresistant cell lines. Prohibitin silencing reduces the overall levels ofprohibitin and results in partial reversal of the resistant phenotype.Unexpectedly, combined reduction of both prohibitin and GST-π results innear complete restoration of paclitaxel sensitivity in vitro. Thisapproach is useful in sensitizing cancer patients whose tumors showresistance to these commonly used drugs.

Briefly, we have seen that in the parental cells (A549 and MES-SA)prohibitin is primarily localized in the mitochondria (See Table 1 for adescription of resistance). In the Taxoid family member resistantvariants (A549TR and MES-SA DX) prohibitin is diffused all over thecell. We have further found that elevated levels of prohibitin ispresent in the cell surface of the Taxoid family member resistant cellsthan in the parental cells.

We have further shown that antagonizing prohibitin and GST-π incombination by simultaneously using siRNA against their mRNA in the drugresistant sublines reverses their paclitaxel resistance significantly.

TABLE 1 Drug Resistant Resistance Parental Cell Line Cell Line RaisedAgainst Cross Resistance Lung Cancer Lung Cancer Taxol Multiple drug(A549) (A549TR) resistant (MDR) Uterine Sarcoma Uterine SarcomaDoxorubicin Multiple Drug (MES-SA) (MES-SA DX) Resistant (MDR) Taxol

TABLE II Proteins Identified in both A549TR and MES-SA DX5 CellsExpression Pattern Cellular Fraction Glutathione-S-Transferase-piOverexpressed Cytoplasm Prohibitin Overexpressed Microsomal Tubulin β-5Overexpressed Cytoplasm Anhexin I Overexpressed Cytoplasm LipocortinUnderexpressed Microsomal

All references cited herein are incorporated herein in their entirety byreference.

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1. A method for predicting the resistance of cancer to a member of thetaxoid family in a subject, the method comprising: a. measuring a levelof prohibitin in a biological sample obtained from a subject; and b.comparing the level measured in step (a) to a standard level, wherein anelevation of the measured level of prohibitin relative to the standardlevel is indicative of resistance to a member of the taxoid family.
 2. Amethod for predicting the resistance of cancer in a subject to a memberof the taxoid family, the method comprising: a. measuring the level ofprohibitin in multiple biological samples obtained from a subjectperiodically over a period time; and b. measuring a change in themeasured level of prohibitin in the biological samples, wherein anelevation of the level of prohibitin is indicative of resistance to amember of the taxoid family.
 3. The method of claim 1 or 2, wherein thesubject had been treated with the member of the taxoid family.
 4. Themethod of claim 1 or 2, wherein the member of the taxoid family ispaclitaxel or docetaxel.
 5. The method of claim 1 or 2, wherein serialmonitoring of the level of prohibitin is performed at least quarterly,at least bimonthly, at least biweekly, at least weekly, at least everythree days or at least daily.
 6. The method of claim 1 or 2, wherein thelevel of prohibitin is measured by measuring the level of prohibitin onthe cell surface of cancer cells in the biological sample.
 7. The methodof claim 1 or 2, wherein the biological sample is selected from thegroup consisting of blood, tissue, serum, plasma, urine, stool,cerebrospinal fluid, nipple aspirates, tumor biopsy, and cell lysate. 8.The method of claim 7, wherein the biological sample is a cell lysateand the level of prohibitin is measured by measuring the level ofprohibitin in the microsomal fraction of the biological sample.
 9. Themethod of claim 7, wherein the biological sample is serum and the levelof prohibitin is determined by measuring the level of prohibitin in theserum.
 10. The method of claim 7, wherein the biological sample is bloodand the level of prohibitin is determined by measuring the level ofprohibitin in platelets of the blood sample.
 11. The method of claim 1or 2, wherein the level of prohibitin protein is measured using anantibody-based binding moiety which specifically binds prohibitin. 12.The method according to claim 11, wherein the antibody-based bindingmoiety is labeled with a detectable label.
 13. The method according toclaim 12, wherein the label is selected from the group consisting of aradioactive label, a hapten label, a fluorescent label, and an enzymaticlabel.
 14. The method according to claim 11, wherein the antibody-basedbinding moiety is an antibody.
 15. The method according to claim 14,wherein the antibody is a monoclonal antibody.
 16. The method of claim 1or 2, wherein the level of prohibitin protein is measured using WesternBlot analysis, Fluorescent activated cell sorting (FACS), enzyme-linkedimmunosorbent assay (ELISA), immunohistochemistry, mass spectrometry,radio-immunoassy, surface plasmon resonance, or immunofluorescence. 17.A method for improving the effectiveness of cancer treatment comprisingmeasuring the level of prohibitin in a biological sample, wherein anelevated level of prohibitin as compared to a standard level indicatesthat the subject is resistant to treatment with a member of the taxoidfamily and is in need of alternative treatment so as to improve theeffectiveness of cancer treatment.
 18. The method of claim 17, whereinthe subject had been treated with the member of the taxoid family. 19.The method of claim 18, wherein the member of the taxoid family ispaclitaxel or docetaxel.
 20. The method of claim 17, wherein serialmonitoring of the level of prohibitin is performed at least quarterly,at least bimonthly, at least biweekly, at least weekly, at least everythree days or at least daily.
 21. The method of claim 17, wherein thelevel of prohibitin is measured by measuring the level of prohibitin onthe cell surface of cancer cells in the biological sample.
 22. Themethod of claim 17, wherein the biological sample is selected from thegroup consisting of blood, tissue, serum, urine, stool, plasma,cerebrospinal fluid, nipple aspirates, tumor biopsy, and cell lysate.23. The method of claim 22, wherein the biological sample is a celllysate and the level of prohibitin is measured by measuring the level ofprohibitin in the microsomal fraction of the biological sample.
 24. Themethod of claim 22, wherein the biological sample is serum and the levelof prohibitin is determined by measuring the level of prohibitin in theserum.
 25. The method of claim 22, wherein the biological sample isblood and the level of prohibitin is determined by measuring the levelof prohibitin in platelets of the blood sample.
 26. The method of claim17, wherein the level of prohibitin protein is measured using anantibody based binding moiety which specifically binds prohibitin. 27.The method according to claim 26, wherein the antibody-based bindingmoiety is labeled with a detectable label.
 28. The method according toclaim 27, wherein the label is selected from the group consisting of aradioactive label, a hapten label, a fluorescent label, and an enzymaticlabel.
 29. The method according to claim 28, wherein the antibody-basedbinding moiety is an antibody.
 30. The method according to claim 29,wherein the antibody is a monoclonal antibody.
 31. The method of claim17, wherein the level of prohibitin protein is measured using WesternBlot analysis, Fluorescent activated cell sorting (FACS), enzyme-linkedimmunosorbent assay (ELISA), surface plasmon resonance,immunohistochemistry, mass spectrometry, radio-immunoassay orimmunofluorescence.
 32. A method of treating cancer comprisingadministering to a subject an inhibitor of prohibitin.
 33. The method ofclaim 32, wherein the cancer is resistant to a taxoid family member. 34.The method of claim 32, further comprising administering a taxoid familymember.
 35. The method of claim 34, wherein the taxoid family member ispaclitaxel.
 36. The method of claim 32, wherein the inhibitor inhibitstranslocation of prohibitin to the cell surface.
 37. The method of claim32, wherein the inhibitor of prohibitin inhibits transcription orexpression of prohibitin.
 38. The method of claim 32, wherein theinhibitor of prohibitin is selected from the group consisting of siRNA,antibody, small molecule, or peptide.
 39. The method of claim 32,further comprising administering glutathione-S-transferase π.
 40. Amethod of treating a cancer in a subject that is resistant to a taxoidfamily member comprising administering to the subject a compoundcomprising an agent that selectively binds prohibitin and a therapeuticagent.
 41. The method of claim 40, wherein the agent that selectivelybinds prohibitin is a peptide having the amino acid sequence of SEQ IDNO:2.
 42. The method of claim 40, wherein agent that selectively bindsprohibitin is an antibody-based binding moiety.
 43. The method of claim40, wherein the therapeutic agent is a drug, a chemotherapeutic agent, aradioisotope, a pro-apoptosis agent, an anti-angiogenic agent, ahormone, a cytokine, a cytotoxic agent, a cytocidal agent, a peptide, aprotein, an antibiotic, an antibody, a Fab fragment of an antibody, ahormone antagonist, and an antigen.
 44. A method to direct treatment ofa subject which comprises having a subject tested for the level ofprohibitin in a biological sample from the subject, wherein a clinicianreviews the results as compared to a standard level of prohibitin, andif the biological sample has an elevated level of prohibitin as comparedto a standard level the clinician directs the subject to be treated withan inhibitor of prohibitin.
 45. A method to direct treatment of asubject which comprises having a subject tested for the level ofprohibitin in a biological sample from the subject, wherein a clinicianreviews the results as compared to a standard level of prohibitin, andif the biological sample has an elevated level of prohibitin as comparedto a standard level the clinician directs the subject to be treated witha compound comprising an agent that selectively binds prohibitin and atherapeutic agent.